Protocol for generating reproducible miniaturized controlled midbrain organoids

Summary Here, we present a protocol for generating miniaturized controlled midbrain organoids (MiCOs) of reproducible size and cellular composition, without a necrotic center. We describe steps for maintaining and passaging human pluripotent stem cells, generating MiCOs using AggreWellTM400, and maintaining them in an EB-Disk360on an orbital shaker, eliminating the need for Matrigel or a spinner flask and preventing organoid fusion. We then detail organoid collection for different endpoint analysis. This protocol is suitable for compound screening and disease modeling studies.


MATERIALS AND EQUIPMENT
hPSC culture and ventral midbrain MiCO culture media hPSC culture media Thaw TeSRä-E8ä 253 Supplement (20 mL) and add into 480 mL of TeSRä-E8ä Basal Medium. Add 2.5 mL Penicillin-Streptomycin (Pen/Strep). Mix thoroughly. Aliquot into 50 mL polypropylene tubes (45 mL/tube) and store at À20 C for up to 6 months. Once the complete TeSRä-E8ä medium is thawed, can be store at 4 C for up to 2 weeks. Do not refreeze thawed media.
Alternatives: Other hPSC culture media such as mTeSR1, mTeSRä Plus or IPS-Brew Medium can be used.

Midbrain MiCOs generation and culture media
Small molecular preparation SB431542 Prepare 50 mM stock in DMSO, aliquot (e.g., in 25 mL) and store at À20 C for up to one year. Prepare 10 mM stock by adding 10 mL of 50 mM SB into 40 mL absolute ethanol and store at À20 C for up to 6 months.

LDN193189
Prepare 1 mM stock in DMSO, aliquot (e.g., in 25 mL) and store at À20 C for up to one year. Prepare 100 mM stock by diluted 10 mL of 1 mM stock with 90 mL of absolute ethanol, store at À20 C for up to 6 months.

CHIR99021
Prepare 1 mM stock in DMSO, aliquot (e.g., in 25 mL) and store at À20 C for up to one year.

SAG
Prepare 200 mM stock in PBS-, aliquot (e.g., in 200 mL) and store at À80 C for up to three years. Thaw aliquot store at 4 C for up to three months.

FGF8b
Prepare 25 ng/mL stock in PBS-with 0.1% BSA solution, aliquot (e.g., in 100 mL) and store at À80 C for up to one year. Thaw aliquot store at 4 C for up to one month.

LM22A4
Prepare 10 mM stock in H2O, aliquot (e.g., in 50 mL) and stored at À20 C for up to one year. Thaw aliquot store at 4 C for up to three months.

L-Ascorbic acid (AA)
Prepare 50 mM stock in H2O, aliquot (e.g., in 500 mL) and stored at À20 C for up to one year. Thaw aliquot store at 4 C for up to three months.

GDNF
Prepare 10 mg/mL stock in PBS-with 0.1% BSA solution, aliquot (e.g., in 50 mL) and store at À20 C for up to one year. Thaw aliquot store at 4 C for up to one month.
cAMP Prepare 50 mM stock solution in H2O, aliquot (e.g., in 500 mL) and stored at À20 C for up to one year. Thaw aliquot store at 4 C for up to three months.

DAPT
Prepare 10 mM stock in DMSO, aliquot (e.g., in 50 mL) and store at À20 C for up to one year. Prepare 1 mM stock by diluted 10 mL of 10 mM stock with 90 mL of absolute ethanol, store at À20 C for up to 6 months.

Y-27632 ROCK inhibitor
Prepare 50 mM stock solution in H20, aliquot (e.g., in 20 mL) and store at À20 C for up to one year. Prepare 10 mM stock solution by diluted 10 mL of 50 mM stock with 40 mL of DMEM, store at 4 C for up to three months. Note: The coated dish(es) can be kept at 4 C for up to 4 weeks, make sure the coated surface does not dehydrate. LN521 coating solution can not be reused.

Timing: 30 min
This step is to grow hPSCs to 75%-80% confluency for starting a midbrain MiCO experiment. hPSCs are cultured under feeder-free condition on Biolaminin (LN521, 0.5 mg/cm 2 in PBS++) coated dishes. We used gentle cell dissociation buffer (0.5 mM EDTA in PBS--) to passage the hPSCs and plated 40,000-50,000 cells on a dish (Ø = 35 mm, 9 cm 2 ) and cultured for 5 days. We normally start this step on a Wednesday, and then the cells will be ready to be used on a Monday to start a vmDA MiCOs generation experiment.
3. Gentle cell dissociation buffer is prepared by adding 10 mL of EDTA (0.5 M) to 10 mL of PBS--to have final 0.5 mM EDTA solution. 4. Pre-warm E8 media to room temperature (20 C-25 C). 5. Aspirate LN521 solution from the pre-coated dish(es) and replace with 1 mL of fresh E8 media. 6. Aspirate the hPSCs culture media and wash the cells one time with PBS-. 7. Aspirate the PBS-, and add 1 mL of gentle cell dissociation buffer.
Note: The incubation time can vary between different cell lines. It also depends on how recently the gentle cell dissociation buffer was prepared. We recommend using the gentle cell dissociation buffer within two weeks.
8. Aspirate the gentle cell dissociation buffer, add 1 mL fresh E8 media. a. Use P1000 pipette to scrape cells off from the dish. b. Pipette the cell suspension once to twice to break the colonies into smaller pieces. c. Collect the cells and transfer them to a 1.5 mL Eppendorf tube. 9. Use a P200 pipette to take out 100 mL cell suspension and transfer to a new 1.5 mL Eppendorf tube. a. Pipette up and down about 20-25 times to have single cell suspension. b. Take out 10 mL for counting the cell density.

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10. Calculate cell number and plate 40,000 cells on the pre-coated LN521 dish with E8 media. Make sure the cells distribute uniformly on the dish(es). 11. Transfer the dish(es) into a 37 C incubator. Media change every two or three days.
Start a midbrain MiCO generation experiment (Day 0)

Timing: 1.5 h
This step is to generate MiCOs from AggreWellä400 24-well plate. We have tested cell numbers ranging from 50 to 1000 per MiCO. And we recommend that 300 to 750 cells per MiCO is a good range for generating MiCOs.
Prepare AggreWellä400 24-well plate 12. Add 500 mL Anti-Adherence Rinsing Solution to the number of wells to be used. 13. Centrifuge 1300 x g for 5 min. 14. Check the microwells under a microscope to ensure that there are no bubbles in wells. If there are any bubbles, repeat step 13. 15. Aspirate Anti-Adherence Rising Solution from the well(s). 16. Rinse the well(s) with pre-warm N2B27 media (2 mL/well). 17. Aspirate N2B27 media and add prewarm N2B27 media (1 mL/well) containing small molecules (Midbrain floor plate patterning media (day0-day9)) and 10 mM ROCK inhibitor. 18. Leave the AggreWellä400 24-well plate in the incubator at 37 C.
Note: A balance plate is needed for step 13.

Prepare hPSCs
19. Under the stereomicroscope, use a glass pipette to scrape off and remove any differentiated cells. 20. Aspirate E8 medium from the dish. 21. Gently wash the cells with 1 mL PBS-. 22. Aspirate the PBS-, add 1 mL of Gentle Cell Dissociation Buffer.
a. Pipette the cell suspension once to twice to break the colonies into smaller pieces. b. Collect the cells and transfer them to a 1.5 mL Eppendorf tube.
Seeding hPSCs on AggreWellä400 24-well plate 25. Use a P200 pipette to take out 100 mL cell suspension from step 24 and transfer to a new 1.5 mL Eppendorf tube. a. Pipette up and down about 20-25 times to have single cell suspension. b. Take out 10 mL for counting the cell density. c. Repeat cell counting and aim for the difference between the two counts is less than 20%. 26. Calculate the volume of cell suspension to be used for seeding. Add the cell suspension to the well of Aggrewellä400 and add N2B27 containing Small Molecules (Midbrain floor plate patterning media (day0-day9)) and 10 mM ROCK inhibitor to achieve a final volume of 2 mL/well. Note: For step 26, the cell suspension volume added should be less than 1 mL. If the cell density is too low, spin down and resuspend to have cell density above 1 million cells/mL. There are approximately 1,200 microwells per well of the AggreWellä400 24-well plate. If desired 500 cells/microwell then seed 600,000 (500*1200) live cells per well (troubleshooting 1).
For a new cell line, cell number titration can be done by seeding different cell amounts per MiCO. For example, if the cell numbers per MiCO are 300, 500, and 750, three wells of AggreWellä400 will be seeded with 360,000 (300*1200), 600,000 (500*1200), and 900,000 (750*1200) live cells, respectively. MiCOs are maintained as described below and sizes of the MiCOs will be measured and necrotic center formation will be tested by Cleaved Caspase-3 staining. The optimized cell amount is achieved when the size of the MiCOs do not exceed 800 mm and no necrotic center is detected.
27. Pipette cells carefully up and down several times without introducing bubbles to the well. 28. Immediately centrifuge at 100 x g for 3 min and check even cell distribution under a microscope. 29. Place the plate in the incubator. This day is day 0 of the culturing period.
Note: For step 28, a balance plate is needed.
Half-medium change for AggreWellä400 24-well plate (Day 2-Day9) Note: The coating solution 5% Pluronic F-127 for step 35 can be stored for at least 8 months at 4 C. The EB-Disk 360 will not stay at the bottom of the well but will be floating in the medium. When samples are taken out from À80 C for sectioning, it is recommended to wait for 20-30 min until the samples reach a temperature of À20 C.

Immunostaining of midbrain MiCO sections
Timing: 2 days

EXPECTED OUTCOMES
Reproducible MiCOs can be maintained with this protocol for up to 150 days. This protocol is an efficient and cost-effect way to maintain and change media for up to 360 MiCOs in one well of a 6-well plate. As shown in Figure 1, 1200 MiCOs can be generated in one well of 24-well plate format in the AggreWell TM 400. MiCOs can be transferred to EB-Disk 360 from day 4 to day 9. From then on, the MiCOs are maintained in the EB-Disk 360 for up to 5 months. The MiCOs remain intact and grow slowly during further differentiation and maturation ( Figure 2) which is key to avoiding the formation of a necrotic core.

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vmDA MiCOs will express floor plate progenitor markers such as LMX1A/FOXA2, as well as caudal midbrain makers OTX2/EN1 on day 16. [2][3][4][5] To confirm the presence of vm dopaminergic neurons, immunostaining with TH and co-staining with FOXA2, LMX1A, and EN1 was performed and showed robust expression at day 50 4,5 . On day 75, majority of TH cells were positive with GIRK2 staining while a small portion of TH cells were positive with CALB staining (Figure 3), which indicating that the protocol generate more A9 neurons compared to A10 neurons. 6,7 TH and MAP2 staining are seen both on the edge and the center of the MiCOs (Figures 3 and 4). The size of the MiCOs did not exceeded 800 mm and most remarkably there was no necrotic center detected at day 75 (Figure 4

) (troubleshooting 5).
Note: It is suggested to do immunostainings on day 16 to make sure the progenitors are patterned correctly (positive staining for FOXA2/LMX1A and OTX2/EN1) before extending the MiCOs for long-term culture. In case of the cell patterning is wrong, stop the experiment and start a new round.
Depending on the downstream analysis, MiCOs can be taken out for different assays such as western blot, dopamine measurement, electrophysiology and so on. For example, MiCOs can be cultured individual in a multi-well plate such as a 96-well plate for drug screening.

LIMITATIONS
This protocol requires the titration of the CHIR concentration for each individual cell line to ensure high efficiency of ventral midbrain NPCs and dopaminergic neurons.
Depending on the experimental set up and downstream analysis, long-term maintenance of the MiCOs are required, which can take months.
It is time consuming when transferring MiCOs from AggreWellä400 to EB-Disk 360 (30-45 min for transferring 360 MiCOs into an EB-Disk 360 ). However, once this step is done, it is very easy to change media and culture/maintain the MiCOs in this format. This format also saves media compared to using a spinner flask culture system and avoids using Matrigel to embed the MiCOs.

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In the MiCO system, we have not looked for the presence of alpha-Synuclein protein aggregates or disease related mechanisms. However, patient-derived human iPSCs differentiated into mesencephalic dopaminergic neurons in organoids have shown evidence of aggregated synuclein and progressive loss of dopaminergic neurons. 8,9 TROUBLESHOOTING Problem 1 Cell number per MiCO is less than 50 or higher than 1000 from a healthy cell line will be difficult to use this protocol.

Potential solution
Cell number per MiCO is optimized to be between 100 to 750 for a non-disease cell line. We have tested lowest cell numbers per MiCO from 50 cells and 100 cells per MiCO. When the cell number is 50 per MiCO, the MiCO is difficult to maintain long-term and challenging to analyze. The lowest cell number per MiCO we tested is 100 cells, and we succeed with maintain these until day 42. (Figure 5A).
For disease cell lines, cell number titration needs to be tested.

Problem 2
There is a risk that MiCOs may break or stick onto the cell strainer during dislodgment and transfer.

Potential solution
Slowly pipette up and down 3-5 times with the 2 mL serological pipette in the AggreWell so that most of the MiCOs are transfered from the AggreWellä400 to the inverted 40 mm cell strainer. Then quickly use PBS++ to rinse the AggreWellä400 to get all of the MiCOs onto the cell strainer and rinse again with PBS++ to avoid MiCOs dehydrated and stick onto the cell strainer. And finally, quickly elute the MiCOs from the cell strainer (need to invert the cell strainer to make sure it is in the correct position) with 2 mL complete media into a clean dish.

Problem 3
MiCOs will stick together when harvesting them in a dish for longer than 1-2 h at room temperature (20 C-25 C) ( Figure 5B).

Potential solution
It is important to harvest one well of an AggreWellTM 400 and transfer the MiCOs to EB-disk360 at a time. If it takes longer than 1 h to transfer, we recommend using 10 mL complete medium in the dish (Ø = 100mm) to help keep the MiCOs separated.

Problem 4
Poor immunostaining result from day 16 samples (MiCOs generated from 100 cells per MiCO on day 0) when 4% PFA fix for 2 h at room temperature (20 C-25 C) ( Figure 5C).

Potential solution
Fixation time needs to be optimized. Since the MiCOs generated from this protocol are less than 1 mm in size, the 4% PFA fixation time needs to be shorten to 20 min at room temperature (20 C-25 C). For other antibodies not mentioned in this protocol, fixation time may need to be optimized.

Problem 5
MiCOs did not show correct patterning with fewer cells positive immunostainings with FOXA2/ LMX1A on day 16.

Potential solution
Start new experiment round with new small molecular aliquots.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mark Denham (mden@dandrite.au.dk).

Materials availability Not applicable.
Data and code availability Not applicable.